1. Field:
This disclosure is concerned generally with an analytical method for determining very small concentrations of a substance in a liquid. More specifically, the disclosure is concerned with an immunoassay for cortisol.
2. Prior Art:
The term immunoassay (IA) refers to methods for determining the presence or concentration of a substance in a fluid which methods require, at some point, the complexation of an antibody and a substance (antigen or hapten) to which the antibody is specific. Since it is known that antibodies to a given substance tend to be very specific to that substance, that specificity has been employed in a variety of immunoassay techniques to determine the presence or concentration of substances which are present in very small quantities in human body fluids such as blood.
Typically, an immunoassay is based on a competition between molecules of a given substance and similar molecules (which have been labeled) for a limited number of complexing sites on antibodies which are specific to the substance (both the unlabeled and labeled forms). After the competition for complexing sites, the complexed materials are separated from the reaction solution and the amount of label associated with the separated materials or the remaining solution is then quantitated. That quantitation can be related to previously prepared standards to determine the presence or amount of the unlabeled substance.
A variety of labels are known and used in immunoassays. For example, fluorogenic materials useful in a fluoroimmunoassay (FIA) are described in U.S. Pat. No. 3,940,475 to Gross. Enzyme labels can be coupled to antibodies or antigens to perform an enzyme immunoassay (EIA) as illustrated in U.S. Pat. No. 3,654,090 to Schuurs et al. Radioisotopes can be incorporated into an antibody or substance (antigen or hapten) to perform a radioimmunoassay (RIA) as described in U.S. Pat. No. 3,555,143 to Axen et al. As used herein, the expression labeled substance, label, marker, tracer, or the equivalent, includes any of those known labels.
As pointed out, an immunoassay requires, at some point, an immunochemical complexation between a substance and its antibody, one of which is labeled. By allowing a suitable incubation period during which labeled substances compete with, complex with, or displace unlabeled substances, and then quantitating the label (e.g. fluorometrically, enzymatically, radiometrically, etc.), it is possible to determine an unknown by known means.
Prior to such quantitation, however, it is necessary to separate the immunochemically complexed products (which must include at least some label) from the surrounding incubation medium (which includes the remaining label). Such separations can be facilitated by providing one of the complexing species in an immobilized (insolubilized) yet active form. For example, it is known that antigenic substances, haptens, or antibodies thereto can be attached to, or incorporated in, various water insoluble carrier materials without substantial loss of biological or immunochemical activity. See, for example, U.S. Pat. No. 3,555,143 (organic supports or carriers) and U.S. Pat. No. 3,652,761 (inorganic carriers). When either of the reactants in an immunoassay is used in such an immobilized form, there is present a solid phase which, when appropriate, can be readily separated (e.g. by centrifugation) for label quantitation. The use of composites comprising antibodies or antigens associated with or immobilized on essentially water insoluble carrier materials is commonly referred to as solid phase immunoassay (SPIA). The use of inorganic siliceous materials as antibody supports in solid phase radioimmunoassays (SPRIA) is described in detail in U.S. Pat. No. 3,975,511 to W. Vann et al.
A major problem in measuring the concentrations of substances present in serum is the interference of other materials which are present, often in much larger concentrations. Very commonly, the interference is due to proteins. It is known that certain interferring proteins sometimes can be inactivated by the use of chemicals known as deblocking agents without significantly affecting the antibody. See, for example, U.S. Pat. No. 3,911,096 to Chopra which discloses the use of deblocking agents such as 8-anilino-1-naphthalene sulfonic acid (ANS) and related compounds for immunoassays (non-SPIA) of throid hormones. Unfortunately, it has been found that when some deblocking agents are used in effective amounts in some IA systems, the antibodies lose part of their activity. This has tended to discourage the use of known deblocking agents in some systems, specifically those systems in which the antibody and interferring protein are inactivated to similar degrees by the deblocking agents.
The desirability of being able to accurately determine cortisol concentrations in human body fluids (e.g. serum) is well known. Cortisol is the major glucocorticoid produced and secreted by the adrenal cortex and its presence affects many body functions. Hence, the determination of cortisol concentration has been found helpful in the diagnosis and treatment of a variety of human ailments. Because the clinically significant concentration range of cortisol is very small (e.g. about 12.5 to 800 ng/ml), cortisol concentrations are commonly determined via immunoassay techniques.
In blood serum, there exist several cortisol binding proteins which can interfere with a cortisol immunoassay. The most commonly cited interfering protein is the protein transcortin (TC). Although transcortin is present in relatively low concentrations, it has a fairly high binding capability for cortisol. Hence, to assure accuracy in any assay for cortisol, it is necessary to eliminate or at least minimize the interfering effects of TC and other cortisol binding proteins, collectively referred to herein as TC.
The use of deblocking agents to assure the release of cortisol from TC had not met with significant success because the deblocking agents also tended to inactivate the cortisol antibodies. Hence, TC removal or inactivation, has tended to require the use of laborious and time-consuming extraction steps or heat denaturation steps. These added steps are not only cumbersome, but also add to the possibility of erroneous results merely because of the added steps required.
Quite surprisingly, we have now developed an extraction-free immunoassay for cortisol which can be performed at room temperature. Details of our assay and preferred methods of performing it are described below.